This invention relates to the field of recombinant DNA technology. In particular, it relates to DNA compounds encoding the enzyme para-nitrobenzyl-esterase (PNB-esterase) from the genus Bacillus. Esters are commonly employed intermediates in the synthesis of cephalosporin and 1-carbacephalosporin antibiotics in free acid form. The preparation of cephalosporins is taught broadly by Chauvette, U.S. Pat. No. 4,064,343. The preparation of 1-carbacephalosporins is taught broadly by Christensen et al., in U.S. Pat. No. 4,226,866, Munroe, in U.S. Pat. No. 4,791,106, and Hirata et al., U.S. Pat. No. 4,708,956.
The PNB-ester function is generally employed to block or protect the acidic carboxylic acid function in the molecule while reactions at other sites of the molecule are carried out. For example, Garbrecht, U.S. Pat. No. 3,632,850, describes the use of the p-nitrobenzyl ester group in the synthesis of cephalexin. In the first step of the synthesis, this ester is cleaved via hydrogenolysis under acidic conditions. In U.S. Pat. No. 3,781,282, Garbrecht describes the de-esterification of p-nitrobenzyl esters of cephalosporins with zinc and acid in an amide-type solvent, such as dimethylformamide. Jackson, U.S. Pat. No. 3,799,924, describes the removal of the p-nitrobenzyl ester group of cephalosporin esters by treatment with sodium or potassium dithionite at a pH above about 7. Hatfield, U.S. Pat. No. 4,091,214, describes a process for de-esterifying esters of cephalosporin compounds which comprises a reductive cleavage employing an inert solvent with zinc and an .alpha.-hydroxycarboxylic acid. Hirata et al., U.S. Pat. No. 4,708,956, describes methods for removal of para-nitrobezyl protecting groups from 1-carbacephalosporin compounds. Attendant with these procedures is the high cost of recycling solvents and the potential problem of pollution caused by organic solvents.
An enzymatic method for removal of PNB blocking groups used in the synthesis of cephalosporin and 1-carbacephalosporin antibiotics would have distinct advantages. Such a reaction, proceeding under mild conditions, could be completed in an aqueous reaction mixture without the use of organic solvents and metallic catalysts. Brannon, U.S. Pat. No. 3,972,774, described a process for the removal of the PNB-ester from cephalosporin esters which comprises reacting the ester with a crude preparation derived from a microorganism of the genus Bacillus. However, the enzyme responsible for this cleavage was not isolated.
A purified PNB-esterase from a microorganism of the genus Bacillus is disclosed in United States Patent Application Attorney Docket No. X-8554, entitled "Purified para-Nitrobenzyl Ester From Bacillus", which was filed on the same day as the present application, and which is incorporated herein by reference. This PNB esterase is a monomer having a molecular weight of about 54,000 daltons. Studies indicate that this PNB-esterase catalyzes the de-esterification of PNB-esters of cephalosporin and 1 -carbacephalosporin compounds to the free acid form. A process for purifying the enzyme from the Bacillus species is also provided by United States Pat. Appln. Attorney Docket No. X-8554. This process comprises a combination of ammonium sulfate fractionation, pH treatment, anion-exchange chromatography, gel filtration, adsorption-desorption chromatography, and affinity chromatography.